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Abstract:

The slow kinetics and low efficiency of reprogramming methods to generate
human induced pluripotent stem cells (iPSCs) impose major limitations on
their utility in biomedical applications. Here we describe a chemical
approach that dramatically improves (>200 fold) the efficiency of iPSC
generation from human fibroblasts, within seven days of treatment. This
will provide a basis for developing safer, more efficient, non-viral
methods for reprogramming human somatic cells.

51. The mixture of claim 48, further comprising an histone deacetylase
(HDAC) inhibitor.

52. A method of inducing non-pluripotent mammalian cells into induced
pluripotent stem cells, comprising: introducing at least one
transcription factor selected from the group consisting of: Oct-3/4,
Klf4, Sox2, and c-Myc into the non-pluripotent cells; and contacting the
non-pluripotent cells with an ALK5 inhibitor, a MEK inhibitor, and a ROCK
inhibitor; under conditions sufficient to induce pluripotent stem cells.

53. The method of claim 52, wherein introducing at least one
transcription factor into the non-pluripotent cells comprises introducing
a polynucleotide encoding the at least one transcription factor into the
non-pluripotent cells.

54. The method of claim 52, wherein introducing at least one
transcription factor into the non-pluripotent cells comprises contacting
the non-pluripotent cells with a polypeptide comprising the amino acid
sequence of the at least one transcription factor polypeptide.

55. The method of claim 52, further comprising contacting the
non-pluripotent cells with a GSK3 inhibitor.

56. The method of claim 52, further comprising contacting the
non-pluripotent cells with an HDAC inhibitor.

57. The method of claim 52, wherein the ROCK inhibitor has the structure:
##STR00019## wherein, L2 is substituted or unsubstituted
C1-C10 alkylene; y is an integer from 0 to 3; z is an integer
from 0 to 5; X is --N═, --CH═ or --CR.sup.5.dbd.; R1 is
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3,
R4 and R5 are independently --CN, --S(O)nR6,
--NR7R8, --C(O)R9, --NR10--C(O)R11,
--NR12--C(O)--OR13, --C(O)NR14R15,
--NR16S(O)2R17, --OR18, --S(O)2NR19,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted heteroaryl, wherein n is an integer from 0
to 2, wherein if z is greater than 1, two R3 moieties are optionally
joined together to form a substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; and
R6, R7, R8, R9, R10, R11, R12,
R13, R14, R15, R16, R17, R18 and R19
are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a
racemate, diastereomer, tautomer, or a geometric isomer thereof, or a
pharmaceutically acceptable salt thereof.

Description:

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §1.119(e)
of U.S. provisional Application No. 61/252,548, filed Oct. 16, 2009, the
contents of which are incorporated by reference in the entirety.

[0003] Although the dangers of genomic insertion of exogenous
reprogramming factors is being overcome, the low efficiency and slow
kinetics of reprogramming continue to present a formidable problem for
ultimate applications of human iPSC. For example, an increase in genetic
or epigenetic abnormalities could occur during the reprogramming process,
where tumor suppressors may be inhibited and oncogenic pathways may be
activated. Though recent studies have reported an improved efficiency of
reprogramming by genetic manipulations (Feng, B. et al., Cell Stem Cell
4, 301-12 (2009)) in addition to the original four factors, such
manipulations typically make the process even more complex and increase
the risk of genetic alterations and tumorigenicity. Thus, there is still
a tremendous need for a safer, easier and more efficient procedure for
human iPSC generation and facilitate identifying and characterizing
fundamental mechanisms of reprogramming.

[0009] In some embodiments, at least 99% of the cells are non-pluripotent
cells. In some embodiments, all or essentially all of the cells are
non-pluripotent cells.

[0010] In some embodiments, the cells are human cells.

[0011] In some embodiments, the TGFβ receptor/ALK5 inhibitor is
SB431542.

[0012] In some embodiments, the MEK inhibitor is PD0325901.

[0013] In some embodiments, the ROCK inhibitor is a compound having the
formula:

##STR00001##

[0014] ring A is a substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted heteroaryl;

[0015] ring B is a substituted or unsubstituted heterocycloalkyl, or
substituted or unsubstituted heteroaryl;

[0016] L1 is --C(O)--NR2-- or --C(O)--NR2--;

[0017] L2 is a bond, substituted or unsubstituted alkylene or
substituted or unsubstituted heteroalkylene; and [0018] R1 and
R2 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl

[0019] In some embodiments, the ROCK inhibitor has the formula:

##STR00002##

wherein, y is an integer from 0 to 3; z is an integer from 0 to 5; X is
--N═, --CH═ or --CR5═; R3, R4 and R5 are
independently CN, S(O)nR6, NR7R8, C(O)R9,
NR10--C(O)R11, NR12--C(O)--OR13,
--C(O)NR14R15, --NR16S(O)2R17, --OR18,
--S(O)2NR19, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n
is an integer from 0 to 2, wherein if z is greater than 1, two R3
moieties are optionally joined together to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl; and R6, R7, R8, R9, R10, R11,
R12, R13, R14, R15, R16, R17, R18 and
R19 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0020] In some embodiments, the ROCK inhibitor has the formula:

##STR00003##

[0021] In some embodiments, the ROCK inhibitor is

##STR00004##

[0022] In some embodiments, the concentration of the inhibitors is
sufficient to improve by at least 10% the efficiency of induction of
non-pluripotent cells in the mixture into induced pluripotent stem cells
when the mixture is submitted to conditions sufficient to induce
conversion of the cells into induced pluripotent stem cells.

[0023] In some embodiments, the mixture further comprises a GSK3 inhibitor
and/or HDAC inhibitor.

[0024] In some embodiments, the polypeptides are selected from Oct-3/4,
Sox2, KLF4 and c-Myc. In some embodiments, the cells are selected from
human cell, non-human animal cells, mouse cells, non-human primates, or
other animal cells.

[0031] In some embodiments, the conditions comprise introducing at least
one exogenous transcription factor into the non-pluripotent cells. In
some embodiments, the at least one exogenous transcription factor is an
Oct polypeptide and the cells are further contacted with a histone
deacetylase (HDAC) inhibitor.

[0032] In some embodiments, the transcription factor is selected from the
group consisting of an Oct polypeptide, a Klf polypeptide, a Myc
polypeptide, and a Sox polypeptide.

[0033] In some embodiments, the method comprises introducing at least two,
three or four exogenous transcription factor into the non-pluripotent
cells, wherein the transcription factors are selected from the group
consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide,
and a Sox polypeptide. In some embodiments, the polypeptides are selected
from Oct-3/4, Sox2, KLF4 and c-Myc. In some embodiments, the cells are
selected from human cell, non-human animal cells, mouse cells, non-human
primates, or other animal cells.

[0034] In some embodiments, the at least one transcription factor is
introduced by introducing a polynucleotide into the non-pluripotent
cells, wherein the polynucleotide encodes the at least one exogenous
transcription factor, thereby expressing the transcription factor(s) in
the cells.

[0035] In some embodiments, the at least one transcription factor is
introduced by contacting an exogenous polypeptide to the non-pluripotent
cells, wherein the polypeptide comprises the amino acid sequence of the
transcription factor, wherein the introduction is performed under
conditions to introduce the polypeptide into the cells. In some
embodiments, the polypeptide comprises an amino acid sequence that
enhances transport across cell membranes.

[0036] In some embodiments, the cells are human cells.

[0037] In some embodiments, the TGFβ receptor/ALK5 inhibitor is
SB431542.

[0038] In some embodiments, the MEK inhibitor is PD0325901

[0039] In some embodiments, the ROCK inhibitor is a compound having the
formula:

##STR00005##

[0040] ring A is a substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted heteroaryl;

[0041] ring B is a substituted or unsubstituted heterocycloalkyl, or
substituted or unsubstituted heteroaryl;

[0042] L1 is --C(O)--NR2-- or --C(O)--NR2--;

[0043] L2 is a bond, substituted or unsubstituted alkylene or
substituted or unsubstituted heteroalkylene; and [0044] R1 and
R2 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0045] In some embodiments, the ROCK inhibitor has the formula:

##STR00006##

wherein, y is an integer from 0 to 3; z is an integer from 0 to 5; X is
--N═, --CH═ or --CR5═; R3, R4 and R5 are
independently CN, S(O)nR6, NR7R8, C(O)R9,
NR10--C(O)R11, NR12--C(O)--OR13,
--C(O)NR14R15, --NR16S(O)2R17, --OR18,
--S(O)2NR19, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n
is an integer from 0 to 2, wherein if z is greater than 1, two R3
moieties are optionally joined together to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl; and R6, R7, R8, R9, R10, R11,
R12, R13, R14, R15, R16, R17, R18 and
R19 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0046] In some embodiments, the ROCK inhibitor has the formula:

##STR00007##

[0047] In some embodiments, the ROCK inhibitor is

##STR00008##

[0048] In some embodiments, the concentration of the inhibitors is
sufficient to improve by at least 10% the efficiency of induction of
non-pluripotent cells in the mixture into induced pluripotent stem cells,
when the mixture is subjected to conditions sufficient to induce
conversion of the cells into induced pluripotent stem cells.

[0049] In some embodiments, the mixture further comprises a GSK3
inhibitor.

[0050] The present invention also provides for kits for inducing
pluripotency in non-pluripotent mammalian cells. In some embodiments, the
kit comprises, [0051] a TGFβ receptor/ALK5 inhibitor; [0052] a MEK
inhibitor; and [0053] a ROCK inhibitor.

[0054] In some embodiments, the TGFβ receptor/ALK5 inhibitor is
SB431542.

[0055] In some embodiments, the MEK inhibitor is PD0325901.

[0056] In some embodiments, the ROCK inhibitor is a compound having the
formula:

##STR00009##

[0057] ring A is a substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted heteroaryl;

[0058] ring B is a substituted or unsubstituted heterocycloalkyl, or
substituted or unsubstituted heteroaryl;

[0059] L1 is --C(O)--NR2-- or --C(O)--NR2--;

[0060] L2 is a bond, substituted or unsubstituted alkylene or
substituted or unsubstituted heteroalkylene; and [0061] R1 and
R2 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl

[0062] In some embodiments, the ROCK inhibitor has the formula:

##STR00010##

wherein, y is an integer from 0 to 3; z is an integer from 0 to 5; X is
--N═, --CH═ or --CR5═; R3, R4 and R5 are
independently CN, S(O)nR6, NR7R8, C(O)R9,
NR10--C(O)R11, NR12--C(O)--OR13,
--C(O)NR14R15, --NR16S(O)2R17, --OR18,
--S(O)2NR19, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n
is an integer from 0 to 2, wherein if z is greater than 1, two R3
moieties are optionally joined together to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl; and R6, R7, R8, R9, R10, R11,
R12, R13, R14, R15, R16, R17, R18 and
R19 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0063] In some embodiments, the ROCK inhibitor has the formula:

##STR00011##

[0064] In some embodiments, the ROCK inhibitor is

##STR00012##

[0065] In some embodiments, the kit further comprises a GSK3 inhibitor
and/or a histone deacetylase (HDAC) inhibitor.

[0066] Other embodiments will be clear from the remainder of this
disclosure.

Definitions

[0067] An "Oct polypeptide" refers to any of the naturally-occurring
members of Octamer family of transcription factors, or variants thereof
that maintain transcription factor activity, similar (within at least
50%, 80%, or 90% activity) compared to the closest related naturally
occurring family member, or polypeptides comprising at least the
DNA-binding domain of the naturally occurring family member, and can
further comprise a transcriptional activation domain. Exemplary Oct
polypeptides include, Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9,
and Oct-11. e.g. Oct3/4 (referred to herein as "Oct4") contains the POU
domain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2,
and uric-86. See, Ryan, A. K. & Rosenfeld, M. G. Genes Dev. 11, 1207-1225
(1997). In some embodiments, variants have at least 85%, 90%, or 95%
amino acid sequence identity across their whole sequence compared to a
naturally occurring Oct polypeptide family member such as to those listed
above or such as listed in Genbank accession number NP--002692.2
(human Oct4) or NP--038661.1 (mouse Oct4). Oct polypeptides (e.g.,
Oct3/4) can be from human, mouse, rat, bovine, porcine, or other animals.
Generally, the same species of protein will be used with the species of
cells being manipulated.

[0068] A "Klf polypeptide" refers to any of the naturally-occurring
members of the family of Kruppel-like factors (Klfs), zinc-finger
proteins that contain amino acid sequences similar to those of the
Drosophila embryonic pattern regulator Kruppel, or variants of the
naturally-occurring members that maintain transcription factor activity
similar (within at least 50%, 80%, or 90% activity) compared to the
closest related naturally occurring family member, or polypeptides
comprising at least the DNA-binding domain of the naturally occurring
family member, and can further comprise a transcriptional activation
domain. See, Dang, D. T., Pevsner, J. & Yang, V. W. Cell Biol. 32,
1103-1121 (2000). Exemplary Klf family members include, Klf1, Klf2, Klf3,
Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14,
Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factors capable
of generating iPS cells in mice, and related genes Klf1 and Klf5 did as
well, although with reduced efficiency. See, Nakagawa, et al., Nature
Biotechnology 26:101-106 (2007). In some embodiments, variants have at
least 85%, 90%, or 95% amino acid sequence identity across their whole
sequence compared to a naturally occurring Klf polypeptide family member
such as to those listed above or such as listed in Genbank accession
number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides
(e.g., Klf1, Klf4, and Klf5) can be from human, mouse, rat, bovine,
porcine, or other animals. Generally, the same species of protein will be
used with the species of cells being manipulated. To the extent a Klf
polypeptide is described herein, it can be replaced with an
estrogen-related receptor beta (Essrb) polypeptide. Thus, it is intended
that for each Klf polypeptide embodiment described herein, a
corresponding embodiment using Essrb in the place of a Klf4 polypeptide
is equally described.

[0069] A "Myc polypeptide" refers any of the naturally-occurring members
of the Myc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol.
Cell Biol. 6:635-645 (2005)), or variants thereof that maintain
transcription factor activity similar (within at least 50%, 80%, or 90%
activity) compared to the closest related naturally occurring family
member, or polypeptides comprising at least the DNA-binding domain of the
naturally occurring family member, and can further comprise a
transcriptional activation domain. Exemplary Myc polypeptides include,
e.g., c-Myc, N-Myc and L-Myc. In some embodiments, variants have at least
85%, 90%, or 95% amino acid sequence identity across their whole sequence
compared to a naturally occurring Myc polypeptide family member, such as
to those listed above or such as listed in Genbank accession number
CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) can be from human,
mouse, rat, bovine, porcine, or other animals. Generally, the same
species of protein will be used with the species of cells being
manipulated.

[0070] A "Sox polypeptide" refers to any of the naturally-occurring
members of the SRY-related HMG-box (Sox) transcription factors,
characterized by the presence of the high-mobility group (HMG) domain, or
variants thereof that maintain transcription factor activity similar
(within at least 50%, 80%, or 90% activity) compared to the closest
related naturally occurring family member, or polypeptides comprising at
least the DNA-binding domain of the naturally occurring family member,
and can further comprise a transcriptional activation domain. See, e.g.,
Dang, D. T., et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000).
Exemplary Sox polypeptides include, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5,
Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17,
Sox18, Sox-21, and Sox30. Sox1 has been shown to yield iPS cells with a
similar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 have also
been shown to generate iPS cells, although with somewhat less efficiency
than Sox2. See, Nakagawa, et al., Nature Biotechnology 26:101-106 (2007).
In some embodiments, variants have at least 85%, 90%, or 95% amino acid
sequence identity across their whole sequence compared to a naturally
occurring Sox polypeptide family member such as to those listed above or
such as listed in Genbank accession number CAA83435 (human Sox2). Sox
polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be from human,
mouse, rat, bovine, porcine, or other animals. Generally, the same
species of protein will be used with the species of cells being
manipulated.

[0072] The term "pluripotent" or "pluripotency" refers to cells with the
ability to give rise to progeny cells that can undergo differentiation,
under the appropriate conditions, into cell types that collectively
demonstrate characteristics associated with cell lineages from all of the
three germinal layers (endoderm, mesoderm, and ectoderm). Pluripotent
stem cells can contribute to all embryonic derived tissues of a prenatal,
postnatal or adult animal. A standard art-accepted test, such as the
ability to form a teratoma in 8-12 week old SCID mice, can be used to
establish the pluripotency of a cell population, however identification
of various pluripotent stem cell characteristics can also be used to
detect pluripotent cells.

[0073] "Pluripotent stem cell characteristics" refer to characteristics of
a cell that distinguish pluripotent stem cells from other cells. The
ability to give rise to progeny that can undergo differentiation, under
the appropriate conditions, into cell types that collectively demonstrate
characteristics associated with cell lineages from all of the three
germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem
cell characteristic. Expression or non-expression of certain combinations
of molecular markers are also pluripotent stem cell characteristics. For
example, human pluripotent stem cells express at least some, and in some
embodiments, all of the markers from the following non-limiting list:
SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin,
UTF-1, Oct4, Rex1, and Nanog. Cell morphologies associated with
pluripotent stem cells are also pluripotent stem cell characteristics.

[0074] As used herein, "non-pluripotent cells" refer to mammalian cells
that are not pluripotent cells. Examples of such cells include
differentiated cells as well as progenitor cells. Examples of
differentiated cells include, but are not limited to, cells from a tissue
selected from bone marrow, skin, skeletal muscle, fat tissue and
peripheral blood. Exemplary cell types include, but are not limited to,
fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts,
and T-cells.

[0075] In some embodiments where an individual is to be treated with the
resulting pluripotent cells, the individual's own non-pluripotent cells
are used to generate pluripotent cells according to the methods of the
invention.

[0077] A "recombinant" polynucleotide is a polynucleotide that is not in
its native state, e.g., the polynucleotide comprises a nucleotide
sequence not found in nature, or the polynucleotide is in a context other
than that in which it is naturally found, e.g., separated from nucleotide
sequences with which it typically is in proximity in nature, or adjacent
(or contiguous with) nucleotide sequences with which it typically is not
in proximity. For example, the sequence at issue can be cloned into a
vector, or otherwise recombined with one or more additional nucleic acid.

[0078] "Expression cassette" refers to a polynucleotide comprising a
promoter or other regulatory sequence operably linked to a sequence
encoding a protein.

[0079] The terms "promoter" and "expression control sequence" are used
herein to refer to an array of nucleic acid control sequences that direct
transcription of a nucleic acid. As used herein, a promoter includes
necessary nucleic acid sequences near the start site of transcription,
such as, in the case of a polymerase II type promoter, a TATA element. A
promoter also optionally includes distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from the
start site of transcription. Promoters include constitutive and inducible
promoters. A "constitutive" promoter is a promoter that is active under
most environmental and developmental conditions. An "inducible" promoter
is a promoter that is active under environmental or developmental
regulation. The term "operably linked" refers to a functional linkage
between a nucleic acid expression control sequence (such as a promoter,
or array of transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs transcription
of the nucleic acid corresponding to the second sequence.

[0080] A "heterologous sequence" or a "heterologous nucleic acid", as used
herein, is one that originates from a source foreign to the particular
host cell, or, if from the same source, is modified from its original
form. Thus, a heterologous expression cassette in a cell is an expression
cassette that is not endogenous to the particular host cell, for example
by being linked to nucleotide sequences from an expression vector rather
than chromosomal DNA, being linked to a heterologous promoter, being
linked to a reporter gene, etc.

[0081] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein to refer to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or double-stranded
form. The term encompasses nucleic acids containing known nucleotide
analogs or modified backbone residues or linkages, which are synthetic,
naturally occurring, and non-naturally occurring, which have similar
binding properties as the reference nucleic acid, and which are
metabolized in a manner similar to the reference nucleotides. Examples of
such analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0083] "Inhibitors," "activators," and "modulators" of expression or of
activity are used to refer to inhibitory, activating, or modulating
molecules, respectively, identified using in vitro and in vivo assays for
expression or activity of a described target protein (or encoding
polynucleotide), e.g., ligands, agonists, antagonists, and their homologs
and mimetics. The term "modulator" includes inhibitors and activators.
Inhibitors are agents that, e.g., inhibit expression or bind to,
partially or totally block stimulation or protease inhibitor activity,
reduce, decrease, prevent, delay activation, inactivate, desensitize, or
down regulate the activity of the described target protein, e.g.,
antagonists. Activators are agents that, e.g., induce or activate the
expression of a described target protein or bind to, stimulate, increase,
open, activate, facilitate, enhance activation or protease inhibitor
activity, sensitize or up regulate the activity of described target
protein (or encoding polynucleotide), e.g., agonists. Modulators include
naturally occurring and synthetic ligands, antagonists and agonists
(e.g., small chemical molecules, antibodies and the like that function as
either agonists or antagonists). Such assays for inhibitors and
activators include, e.g., applying putative modulator compounds to cells
expressing the described target protein and then determining the
functional effects on the described target protein activity, as described
above. Samples or assays comprising described target protein that are
treated with a potential activator, inhibitor, or modulator are compared
to control samples without the inhibitor, activator, or modulator to
examine the extent of effect. Control samples (untreated with modulators)
are assigned a relative activity value of 100%. Inhibition of a described
target protein is achieved when the activity value relative to the
control is about 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of
the described target protein is achieved when the activity value relative
to the control is 110%, optionally 150%, optionally 200, 300%, 400%,
500%, or 1000-3000% or more higher.

[0084] Where chemical substituent groups are specified by their
conventional chemical formulae, written from left to right, they equally
encompass the chemically identical substituents that would result from
writing the structure from right to left, e.g., --CH2O-- is
equivalent to --OCH2--.

[0085] The term "alkyl," by itself or as part of another substituent,
means, unless otherwise stated, a straight (i.e., unbranched) or branched
chain, or combination thereof, which may be fully saturated, mono- or
polyunsaturated and can include di- and multivalent radicals, having the
number of carbon atoms designated (i.e., C1-C10 means one to
ten carbons). Examples of saturated hydrocarbon radicals include, but are
not limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,
cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,
n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is
one having one or more double bonds or triple bonds. Examples of
unsaturated alkyl groups include, but are not limited to, vinyl,
2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers.

[0086] The term "alkylene" by itself or as part of another substituent
means a divalent radical derived from an alkyl, as exemplified, but not
limited, by --CH2CH2CH2CH2--. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those groups
having 10 or fewer carbon atoms being exemplified in the present
invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl
or alkylene group, generally having eight or fewer carbon atoms.

[0087] The term "heteroalkyl," by itself or in combination with another
term, means, unless otherwise stated, a stable straight or branched
chain, or cyclic hydrocarbon radical, or combinations thereof, consisting
of at least one carbon atoms and at least one heteroatom selected from
the group consisting of O, N, P, Si and S, and wherein the nitrogen and
sulfur atoms may optionally be oxidized and the nitrogen heteroatom may
optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be
placed at any interior position of the heteroalkyl group or at the
position at which the alkyl group is attached to the remainder of the
molecule. Examples include, but are not limited to,
--CH2--CH2--O--CH3, --CH2--CH2--NH--CH3,
--CH2--CH2--N(CH3)--CH3,
--CH2--S--CH2--CH3, --CH2--CH2,
--S(O)--CH3, --CH2--CH2--S(O)2--CH3,
--CH═CH--O--CH3, --Si(CH3)3,
--CH2--CH═N--OCH3, --CH═CH--N(CH3)--CH3,
O--CH3, --O--CH2--CH3, and --CN. Up to two heteroatoms may
be consecutive, such as, for example, --CH2--NH--OCH3 and
--CH2--O--Si(CH3)3. Similarly, the term "heteroalkylene"
by itself or as part of another substituent means a divalent radical
derived from heteroalkyl, as exemplified, but not limited by,
--CH2--CH2--S--CH2--CH2-- and
--CH2--S--CH2--CH2--NH--CH2--. For heteroalkylene
groups, heteroatoms can also occupy either or both of the chain termini
(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and
the like). Still further, for alkylene and heteroalkylene linking groups,
no orientation of the linking group is implied by the direction in which
the formula of the linking group is written. For example, the formula
--C(O)2R'-- represents both --C(O)2R'-- and --R'C(O)2--.
As described above, heteroalkyl groups, as used herein, include those
groups that are attached to the remainder of the molecule through a
heteroatom, such as --C(O)R', --C(O)NR', --NR'R'', --OR', --SR', and/or
--SO2R'. Where "heteroalkyl" is recited, followed by recitations of
specific heteroalkyl groups, such as --NR'R'' or the like, it will be
understood that the terms heteroalkyl and --NR'R'' are not redundant or
mutually exclusive. Rather, the specific heteroalkyl groups are recited
to add clarity. Thus, the term "heteroalkyl" should not be interpreted
herein as excluding specific heteroalkyl groups, such as --NR'R'' or the
like.

[0088] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in
combination with other terms, represent, unless otherwise stated, cyclic
versions of "alkyl" and "heteroalkyl", respectively. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of the molecule. Examples of
cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,
1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of
heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and
"heterocycloalkylene" refer to a divalent radical derived from cycloalkyl
and heterocycloalkyl, respectively.

[0089] The terms "halo" or "halogen," by themselves or as part of another
substituent, mean, unless otherwise stated, a fluorine, chlorine,
bromine, or iodine atom. Additionally, terms such as "haloalkyl," are
meant to include monohaloalkyl and polyhaloalkyl. For example, the term
"halo(C1-C4)alkyl" is mean to include, but not be limited to,
trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and
the like.

[0090] The term "aryl" means, unless otherwise stated, a polyunsaturated,
aromatic, hydrocarbon substituent which can be a single ring or multiple
rings (preferably from 1 to 3 rings) which are fused together or linked
covalently. The term "heteroaryl" refers to aryl groups (or rings) that
contain from one to four heteroatoms selected from N, O, and S, wherein
the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen
atom(s) are optionally quaternized. A heteroaryl group can be attached to
the remainder of the molecule through a carbon or heteroatom.
Non-limiting examples of aryl and heteroaryl groups include phenyl,
1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,
2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,
4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,
1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl,
and 6-quinolyl. Substituents for each of the above noted aryl and
heteroaryl ring systems are selected from the group of acceptable
substituents described below. "Arylene" and "heteroarylene" refers to a
divalent radical derived from a aryl and heteroaryl, respectively.

[0091] For brevity, the term "aryl" when used in combination with other
terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and
heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to
include those radicals in which an aryl group is attached to an alkyl
group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including
those alkyl groups in which a carbon atom (e.g., a methylene group) has
been replaced by, for example, an oxygen atom (e.g., phenoxymethyl,
2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0092] The term "oxo" as used herein means an oxygen that is double bonded
to a carbon atom.

[0093] The term "alkylsulfonyl" as used herein means a moiety having the
formula --S(O2)--R', where R' is an alkyl group as defined above. R' may
have a specified number of carbons (e.g. "C1-C4 alkylsulfonyl").

[0094] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and
"heteroaryl") are meant to include both substituted and unsubstituted
forms of the indicated radical. Exemplary substituents for each type of
radical are provided below.

[0095] Substituents for the alkyl and heteroalkyl radicals (including
those groups often referred to as alkylene, alkenyl, heteroalkylene,
heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and
heterocycloalkenyl) can be one or more of a variety of groups selected
from, but not limited to: --OR', ═O, ═NR', ═N--OR', --NR'R'',
--SR', -halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O)2R, --NR--C(NR'R''R''')═NR'''',
--NR--C(NR'R'')═NR''', --S(O)R', --S(O)2R', --S(O)2NR'R'',
--NRSO2R', --CN and --NO2 in a number ranging from zero to
(2m'+1), where m' is the total number of carbon atoms in such radical.
R', R'', R''' and R'''' each preferably independently refer to hydrogen,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),
substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or
arylalkyl groups. When a compound of the invention includes more than one
R group, for example, each of the R groups is independently selected as
are each R', R'', R''' and R'''' groups when more than one of these
groups is present. When R' and R'' are attached to the same nitrogen
atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-,
or 7-membered ring. For example, --NR'R'' is meant to include, but not be
limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion
of substituents, one of skill in the art will understand that the term
"alkyl" is meant to include groups including carbon atoms bound to groups
other than hydrogen groups, such as haloalkyl (e.g., --CF3 and
--CH2CF3) and acyl (e.g., --C(O)CH3, --C(O)CF3,
--C(O)CH2OCH3, and the like).

[0096] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: halogen, --OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''', --NR''C(O)2R',
--NR--C(NR'R''R''')═NR'''', --NR--C(NR'R'')═NR''', --S(O)R',
--S(O)2R', --S(O)2NR'R'', --NRSO2R', --CN and --NO2,
--R', --N3, --CH(Ph)2, fluoro(C1-C4)alkoxy, and
fluoro(C1-C4)alkyl, in a number ranging from zero to the total
number of open valences on the aromatic ring system; and where R', R'',
R''' and R'''' are preferably independently selected from hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and
substituted or unsubstituted heteroaryl. When a compound of the invention
includes more than one R group, for example, each of the R groups is
independently selected as are each R', R'', R''' and R'''' groups when
more than one of these groups is present.

[0097] Two of the substituents on adjacent atoms of the aryl or heteroaryl
ring may optionally form a ring of the formula
-T-C(O)--(CRR')q--U--, wherein T and U are independently --NR--,
--O--, --CRR'-- or a single bond, and q is an integer of from 0 to 3.
Alternatively, two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of the
formula -A-(CH2)r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O)2--,
--S(O)2NR'-- or a single bond, and r is an integer of from 1 to 4.
One of the single bonds of the new ring so formed may optionally be
replaced with a double bond. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be replaced
with a substituent of the formula --(CRR')s--X'--(C''R''')d--,
where s and d are independently integers of from 0 to 3, and X' is --O--,
--NR'--, --S--, --S(O)--, --S(O)2--, or --S(O)2NR'--. The
substituents R, R', R'' and R''' are preferably independently selected
from hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, and substituted or unsubstituted
heteroaryl.

[0098] As used herein, the term "heteroatom" or "ring heteroatom" is meant
to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and
silicon (Si).

[0106] A "size-limited substituent" or "size-limited substituent group,"
as used herein means a group selected from all of the substituents
described above for a "substituent group," wherein each substituted or
unsubstituted alkyl is a substituted or unsubstituted C1-C20
alkyl, each substituted or unsubstituted heteroalkyl is a substituted or
unsubstituted 2 to 20 membered heteroalkyl, each substituted or
unsubstituted cycloalkyl is a substituted or unsubstituted
C4-C8 cycloalkyl, and each substituted or unsubstituted
heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered
heterocycloalkyl.

[0107] A "lower substituent" or "lower substituent group," as used herein
means a group selected from all of the substituents described above for a
"substituent group," wherein each substituted or unsubstituted alkyl is a
substituted or unsubstituted C1-C8 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8
membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a
substituted or unsubstituted C5-C7 cycloalkyl, and each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 5 to 7 membered heterocycloalkyl.

[0108] The term "pharmaceutically acceptable salts" is meant to include
salts of the active compounds which are prepared with relatively nontoxic
acids or bases, depending on the particular substituents found on the
compounds described herein. When compounds of the present invention
contain relatively acidic functionalities, base addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired base, either neat or in a suitable inert
solvent. Examples of pharmaceutically acceptable base addition salts
include sodium, potassium, calcium, ammonium, organic amino, or magnesium
salt, or a similar salt. When compounds of the present invention contain
relatively basic functionalities, acid addition salts can be obtained by
contacting the neutral form of such compounds with a sufficient amount of
the desired acid, either neat or in a suitable inert solvent. Examples of
pharmaceutically acceptable acid addition salts include those derived
from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,
monohydrogencarbonic, phosphoric, monohydrogenphosphoric,
dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or
phosphorus acids and the like, as well as the salts derived from
relatively nontoxic organic acids like acetic, propionic, isobutyric,
maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,
phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino acids
such as arginate and the like, and salts of organic acids like glucuronic
or galactunoric acids and the like (see, for example, Berge et al.,
"Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66,
1-19). Certain specific compounds of the present invention contain both
basic and acidic functionalities that allow the compounds to be converted
into either base or acid addition salts.

[0109] Thus, the compounds of the present invention may exist as salts
with pharmaceutically acceptable acids. The present invention includes
such salts. Examples of such salts include hydrochlorides, hydrobromides,
sulfates, methanesulfonates, nitrates, maleates, acetates, citrates,
fumarates, tartrates (eg (+)-tartrates, (-)-tartrates or mixtures thereof
including racemic mixtures, succinates, benzoates and salts with amino
acids such as glutamic acid. These salts may be prepared by methods known
to those skilled in the art.

[0110] The neutral forms of the compounds are preferably regenerated by
contacting the salt with a base or acid and isolating the parent compound
in the conventional manner. The parent form of the compound differs from
the various salt forms in certain physical properties, such as solubility
in polar solvents.

[0111] In addition to salt forms, the present invention provides
compounds, which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of the
present invention. Additionally, prodrugs can be converted to the
compounds of the present invention by chemical or biochemical methods in
an ex vivo environment. For example, prodrugs can be slowly converted to
the compounds of the present invention when placed in a transdermal patch
reservoir with a suitable enzyme or chemical reagent.

[0112] Certain compounds of the present invention can exist in unsolvated
forms as well as solvated forms, including hydrated forms. In general,
the solvated forms are equivalent to unsolvated forms and are encompassed
within the scope of the present invention. Certain compounds of the
present invention may exist in multiple crystalline or amorphous forms.
In general, all physical forms are equivalent for the uses contemplated
by the present invention and are intended to be within the scope of the
present invention.

[0113] Certain compounds of the present invention possess asymmetric
carbon atoms (optical centers) or double bonds; the racemates,
diastereomers, tautomers, geometric isomers and individual isomers are
encompassed within the scope of the present invention. The compounds of
the present invention do not include those which are known in the art to
be too unstable to synthesize and/or isolate.

[0114] The compounds of the present invention may also contain unnatural
proportions of atomic isotopes at one or more of the atoms that
constitute such compounds. For example, the compounds may be radiolabeled
with radioactive isotopes, such as for example tritium (3H),
iodine-125 (125I) or carbon-14 (14C). All isotopic variations
of the compounds of the present invention, whether radioactive or not,
are encompassed within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] FIG. 1. Compound treatment for seven days is sufficient to induce
pluripotent stem cells from human fibroblasts transduced with the four
reprogramming factors. (a) Timeline for human iPSC induction using
combined SB431542 and PD0325901 treatment along with 4TFs. Treatment
began with cell re-seeding at day 7 after 4TF transduction and was
maintained for 7 days. (b) Staining for ALP.sup.+ colonies that emerged
in the untreated (left) or 2 compound-treated (right) cultures within
seven days. (c) RT-PCR showing elevated endogenous mRNA expression of
pluripotency markers OCT4 and NANOG in 2 compound-treated cultures. (d)
TRA-1-81 staining at day 14 without (left) or with (right) 2 compound
treatment. (e) The numbers of NANOG.sup.+ colonies at day 14 under
different treatment conditions are plotted. (f) Typical staining for
hESC-specific markers (NANOG and SSEA4) exhibited by D14 iPSCs. Scale
bars, 50 μm in (d & f).

[0132] The present invention is based on the surprising discovery that a
combination of an ALK5 inhibitor, a MEK inhibitor, and a ROCK inhibitor
greatly improves efficiency of induction of pluripotency in
non-pluripotent mammalian cells transformed with four transcription
factors. Accordingly, the present invention provides for methods of
inducing pluripotency in non-pluripotent mammalian cells wherein the
method comprises contacting the non-pluripotent cells with at least a
TGFβ receptor/ALK5 inhibitor, preferably in combination with a
MEK/ERK pathway inhibitor, and in particular embodiments, a Rho
GTPase/ROCK inhibitor.

[0135] In view of the data herein showing the effect of inhibiting ALK5,
it is believed that inhibition of the TGFβ/activin pathway will have
similar effects. Thus, any inhibitor (e.g., upstream or downstream) of
the TGFβ/activin pathway can be used in combination with, or instead
of, ALK5 inhibitors as described in each paragraph herein. Exemplary
TGFβ/activin pathway inhibitors include but are not limited to:
TGFβ receptor inhibitors, inhibitors of SMAD 2/3 phosphorylation,
inhibitors of the interaction of SMAD 2/3 and SMAD 4, and
activators/agonists of SMAD 6 and SMAD 7. Furthermore, the
categorizations described below are merely for organizational purposes
and one of skill in the art would know that compounds can affect one or
more points within a pathway, and thus compounds may function in more
than one of the defined categories.

[0138] Inhibitors of the interaction of SMAD 2/3 and smad4 can include
antibodies to, dominant negative variants of and antisense nucleic acids
that target SMAD2, SMAD3 and/or smad4. Specific examples of inhibitors of
the interaction of SMAD 2/3 and SMAD4 include but are not limited to
Trx-SARA, Trx-xFoxH1b and Trx-Lef1. (See, e.g., Cui, et al., Oncogene
24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell,
17:3819-3831 (2006).)

[0139] Activators/agonists of SMAD 6 and SMAD 7 include but are not
limited to antibodies to, dominant negative variants of and antisense
nucleic acids that target SMAD 6 or SMAD 7. Specific examples of
inhibitors include but are not limited to smad7-as PTO-oligonucleotides.
See, e.g., Miyazono, et al., U.S. Pat. No. 6,534,476, and Steinbrecher,
et al., US2005119203, both incorporated herein by reference.

[0140] Those of skill will appreciate that the concentration of the
TGFβ receptor/ALK5 inhibitor will depend on which specific inhibitor
is used. Generally, the concentration of a TGFβ receptor/ALK5
inhibitor in a cell culture will be in the range of IC20-IC100 (i.e.,
concentrations in which 20% inhibition to 100% inhibition in cells is
achieved. For example, SB432542 would be used at 0.5-10 μM, optimally
around 1-5 μM. In certain embodiments, a combination of two or more
different TGFβ receptor/ALK5 inhibitors can be used.

III. MEK/ERK Pathway Inhibitors

[0141] The MEK/ERK pathway refers to the MEK and ERK serine/threonine
kinases that make up part of a signal transduction pathway. Generally,
activated Ras activates the protein kinase activity of RAF kinase. RAF
kinase phosphorylates and activates MEK, which in turn phosphorylates and
activates a mitogen-activated protein kinase (MAPK). MAPK was originally
called "extracellular signal-regulated kinases" (ERKs) and
microtubule-associated protein kinase (MAPK). Thus, "ERK" and "MAPK" are
used synonymously.

[0142] MEK/ERK pathway inhibitors refer to inhibitors of either MEK or ERK
that are part of the Raf/MEK/ERK pathway. Because the inventors have
found that MEK inhibitors are effective in improving induction of iPSCs,
and because MEK directly controls ERK activity, it is believed that MEK
inhibitors as described for the present invention, can be replaced with
an ERK inhibitor as desired.

[0146] Those of skill will appreciate that the concentration of the
MEK/ERK pathway inhibitor will depend on which specific inhibitor is
used. In particular embodiments, a combination of two or more different
MEK/ERK pathway inhibitors can be used.

IV. Rho GTPase/ROCK Inhibitors

[0147] The present invention provides for uses and compositions comprising
inhibitors of the Rho-GTPase/ROCK pathway. The pathway includes the
downstream protein Myosin II, which is further downstream of ROCK
(Rho-ROCK-Myosin II forms the pathway/axis). Thus, one can use any or all
of a Rho GTPase inhibitor, a ROCK inhibitor, or a Myosin II inhibitor to
achieve the effects described herein. Those of skill will appreciate that
the concentration of the Rho-GTPase/ROCK pathway inhibitor will depend on
which specific inhibitor is used. In additional embodiments, a
combination of two or more different Rho-GTPase/ROCK pathway inhibitors
can be used.

[0148] Any Rho GTPase should be effective in the methods and compositions
of the invention. Inhibitors of Rho GTPase can include antibodies that
bind, dominant negative variants of, and siRNA, microRNA, antisense
nucleic acids, and other polynucleotides that target Rho GTPase. An
exemplary Rho GTPase inhibitor is Clostridium botulinum C3 toxin.

[0149] Any Myosin II inhibitor should be effective in the methods and
compositions of the invention. Inhibitors of Myosin II can include
antibodies that bind, dominant negative variants of, and siRNA, microRNA,
antisense nucleic acids, and other polynucleotides that target Myosin II.
An exemplary Myosin II inhibitor is blebbistatin. The inventors have
found that blebbistatin can be substituted for SB431542 (an ALK5
inhibitor), albeit with a reduced effect, in the mixtures and methods
described in the example section. Other inhibitors include but are not
limited to those described in U.S. Pat. No. 7,585,844.

In Formula (I), ring A is a substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl. Ring B is
a substituted or unsubstituted heterocycloalkyl, or substituted or
unsubstituted heteroaryl.

[0156] L1 is --C(O)--NR2-- or --NR2--C(O)--. L2 is a
bond, substituted or unsubstituted alkylene or substituted or
unsubstituted heteroalkylene.

[0158] In some embodiments, ring A is a substituted or unsubstituted aryl.
Ring A may also be a substituted or unsubstituted phenyl.

[0159] In other embodiments, ring B is a substituted or unsubstituted
heterocycloalkyl, or substituted or unsubstituted heteroaryl. Ring B may
also be a substituted or unsubstituted heteroaryl. In still other
embodiments, ring B is a substituted or unsubstituted pyrazolyl,
substituted or unsubstituted furanyl, substituted or unsubstituted
imidazolyl, substituted or unsubstituted isoxazolyl, substituted or
unsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl,
substituted or unsubstituted pyrrolyl, substituted or unsubstituted
pyridyl, substituted or unsubstituted pyrimidyl, substituted or
unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl,
substituted or unsubstituted triazolyl, substituted or unsubstituted
thienyl, substituted or unsubstituted dihydrothieno-pyrazolyl,
substituted or unsubstituted thianaphthenyl, substituted or unsubstituted
carbazolyl, substituted or unsubstituted benzothienyl, substituted or
unsubstituted benzofuranyl, substituted or unsubstituted indolyl,
substituted or unsubstituted quinolinyl, substituted or unsubstituted
benzotriazolyl, substituted or unsubstituted benzothiazolyl, substituted
or unsubstituted benzooxazolyl, substituted or unsubstituted
benzimidazolyl, substituted or unsubstituted isoquinolinyl, substituted
or unsubstituted isoindolyl, substituted or unsubstituted acridinyl,
substituted or unsubstituted benzoisazolyl, or substituted or
unsubstituted dimethylhydantoin.

[0160] L2 may be substituted or unsubstituted C1-C10 alkyl.
In some embodiments, L2 is unsubstituted C1-C10 alkyl.
L2 may also be substituted or unsubstituted methylene (e.g.
unsubstituted methylene).

[0161] R2 may be hydrogen. R1 may be hydrogen or unsubstituted
C1-C10 alkyl. In some embodiments, R1 is simply hydrogen.

[0162] In some embodiments of Formula (I), ring A is substituted or
unsubstituted aryl, ring B is substituted or unsubstituted heteroaryl,
R1 is hydrogen, and L2 is unsubstituted C1-C10 alkyl.

[0163] In another embodiment, the ROCK inhibitor has the formula:

##STR00014##

In Formula (II), y is an integer from 0 to 3 and z is an integer from 0
to 5. X is --N═, --CH═ or --CR4═. R1 and L2
are as defined above in the definitions of Formula (I).

[0164] R3, R4 and R5 are independently --CN,
--S(O)nR6, --NR7R8, --C(O)R9,
--NR10--C(O)R11, --NR12--C(O)--OR13,
--C(O)NR14R15, --NR16S(O)2R17, --OR18,
--S(O)2NR19, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n
is an integer from 0 to 2, wherein if z is greater than 1, two R3
moieties are optionally joined together to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl.

[0166] In some embodiments, L2 is substituted or unsubstituted
C1-C10 alkyl. L2 may also be unsubstituted
C1-C10 alkyl. Alternatively, L2 is substituted or
unsubstituted methylene (e.g. unsubstituted methylene).

[0167] In other embodiments, X is --N═ or --CH═. The symbol z may
be 2. In still other embodiments, two R3 moieties at adjacent
vertices are joined together to from a substituted or unsubstituted
heterocycloalkyl. The symbol z may also be 1. The symbol y may be 0 or 1.
R3 may be --OR18. R18 may be hydrogen or unsubstituted
C1-C10 alkyl.

[0168] In some embodiments, L2 is substituted or unsubstituted
methylene (e.g. substituted methylene), X is --N═ or --CH═,
R1 is hydrogen, and y and z are 0.

[0171] Those of skill will appreciate that the concentration of the GSK3
inhibitor will depend on which specific inhibitor is used. In certain
embodiments, a combination of two or more different GSK3 inhibitors can
be used.

VI. Methods of Inducing Pluripotency

[0172] To date, a large number of different methods and protocols have
been established for inducing non-pluripotent mammalian cells into
induced pluripotent stem cells (iPSCs). It is believed that the agents
described herein can be used in combination with essentially any protocol
for generating iPSCs and thereby improve the efficiency of the protocol.
Thus, the present invention provides for incubation of non-pluripotent
cells with at least a TGFβ receptor/ALK5 inhibitor, preferably in
combination with a MEK/ERK pathway inhibitor, and in particular
embodiments, a Rho GTPase/ROCK inhibitor in combination with any protocol
for generating iPSCs. A selection of protocols is described below and
each is believed to be combinable with the agents of the invention to
improve efficiency of the protocol.

[0173] The improvement in efficiency of an iPSC generation protocol will
depend on the protocol and which agents of the invention are used. In
some embodiments, the efficiency is improved by at least 10%, 20%, 50%,
75%, 100%, 150%, 200%, 300% or more compared to the same protocol without
inclusion of the agents of the invention (i.e., TGFβ receptor/ALK5
inhibitor, MEK/ERK pathway inhibitor and Rho GTPase/ROCK inhibitor).
Efficiency is measured with regard to improvement of the number of iPSCs
generated in a particular time frame or the speed by which iPSCs are
generated.

[0175] As noted above, while the original protocol involved introduction
of four transcription factors into non-pluripotent cells, it has been
more recently discovered that some transcription factors can be omitted.
Thus, in some embodiments, the protocols involves introducing one, two or
three of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a
Sox polypeptide to non-pluripotent cells under conditions that allow for
the non-pluripotent cells to become iPSCs. For example, each of Maherali
and Konrad Hochedlinger, "Tgfβ Signal Inhibition Cooperates in the
Induction of iPSCs and Replaces Sox2 and cMyc" Current Biology (2009) and
WO/2009/117439 describe protocols that do not require all four
transcription factors to induce pluripotency. Moreover, the inventors
have found that iPSCs can be generated by introducing Oct4 alone into
cells and incubating the cells with a TGFβ receptor/ALK5 inhibitor,
a MEK/ERK pathway inhibitor, a Rho GTPase/ROCK inhibitor, and a histone
deacetylase (HDAC) inhibitor. For example, introduction of exogenous Oct4
into mammalian cells, in the presence of a sufficient amount of SB431542,
PD0325901, Tzv, and valproic acid or sodium butyrate, successfully
generated iPSC cells.

[0178] Moreover, recently, it has been shown that transcription factors
can be delivered as exogenous protein to non-pluripotent cells, to
generate iPSCs. See, e.g., WO/2009/117439; Zhou et al., Cell Stem Cell
4:381-384 (2009). One can introduce an exogenous polypeptide (i.e., a
protein provided from outside the cell and/or that is not produced by the
cell) into the cell by a number of different methods that do not involve
introduction of a polynucleotide encoding the polypeptide. Thus, in some
embodiments, non-pluripotent cells are contacted with a TGFβ
receptor/ALK5 inhibitor, preferably in combination with a MEK/ERK pathway
inhibitor, and in particular embodiments, a Rho GTPase/ROCK inhibitor and
one or more exogenous transcription factor proteins, e.g., one, two,
three or all four of an Oct polypeptide, a Klf polypeptide, a Myc
polypeptide, and a Sox polypeptide.

[0179] A variety of ways have been described for introducing the relevant
protein factors into the target cells. In one embodiment, introduction of
a polypeptide into a cell can comprise introduction of a polynucleotide
comprising one or more expression cassettes into a cell and inducing
expression, thereby introducing the polypeptides into the cell by
transcription and translation from the expression cassette.

[0180] Alternatively, one or more proteins can simply be cultured in the
presence of target cells under conditions to allow for introduction of
the proteins into the cell. See, e.g., Zhou H et al., Generation of
induced pluripotent stem cells using recombinant proteins. Cell Stem
Cell. 2009 May 8; 4(5):381-4. In some embodiments, the exogenous proteins
comprise the transcription factor polypeptide of interest linked (e.g.,
linked as a fusion protein or otherwise covalently or non-covalently
linked) to a polypeptide that enhances the ability of the transcription
factor to enter the cell (and in some embodiments the cell nucleus).

[0181] Examples of polypeptide sequences that enhance transport across
membranes include, but are not limited to, the Drosophila homeoprotein
antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:
1121-34,1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-8,1991;
Le Roux et al., Proc. Natl. Acad. Sci. USA, 90: 9120-4,1993), the herpes
simplex virus structural protein VP22 (Elliott and O'Hare, Cell 88:
223-33,1997); the HIV-1 transcriptional activator TAT protein (Green and
Loewenstein, Cell 55: 1179-1188, 1988; Frankel and Pabo, Cell 55: 1
289-1193, 1988); delivery enhancing transporters such as described in
U.S. Pat. No. 6,730,293 (including but not limited to an peptide sequence
comprising at least 7-25 contiguous arginines); and commercially
available Penetratin® 1 peptide, and the Diatos Peptide Vectors
("DPVs") of the Vectocell® platform available from Daitos S. A. of
Paris, France. See also, WO/2005/084158 and WO/2007/123667 and additional
transporters described therein. Not only can these proteins pass through
the plasma membrane but the attachment of other proteins, such as the
transcription factors described herein, is sufficient to stimulate the
cellular uptake of these complexes.

[0182] In some embodiments, the transcription factor polypeptides
described herein are exogenously introduced as part of a liposome, or
lipid cocktail such as commercially available Fugene6 and Lipofectamine).
In another alternative, the transcription factor proteins can be
microinjected or otherwise directly introduced into the target cell.

[0183] As discussed in the Examples of WO/2009/117439, incubation of cells
with the transcription factor polypeptides of the invention for extended
periods is toxic to the cells. Therefore, the present invention provides
for intermittent incubation of non-pluripotent mammalian cells with one
or more of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide,
and/or a Sox polypeptide, with intervening periods of incubation of the
cells in the absence of the one or more polypeptides. In some
embodiments, the cycle of incubation with and without the polypeptides
can be repeated for 2, 3, 4, 5, 6, or more times and is performed for
sufficient lengths of time (i.e., the incubations with and without
proteins) to achieve the development of pluripotent cells. Various agents
(e.g., MEK/ERK pathway inhibitor and/or GSK3 inhibitor and/or
TGFbeta/ALK5 inhibitor and/or Rho GTPase/ROCK pathway inhibitor) can be
included to improve efficiency of the method.

[0184] The various inhibitors (e.g., TGFβ receptor/ALK5 inhibitor,
MEK/ERK pathway inhibitor, and in particular embodiments, Rho GTPase/ROCK
inhibitor, and/or GSK3 inhibitor, etc.) can be contacted to
non-pluripotent cells either prior to, simultaneous with, or after
delivery of, programming transcription factors (for example, delivered
via expression cassette or as proteins). For convenience, the day the
reprogramming factors are delivered is designated "day 1". In some
embodiments, the inhibitors are contacted to cells in aggregate (i.e., as
a "cocktail") at about days 3-7 and continued for 7-14 days.
Alternatively, in some embodiments, the cocktail is contacted to the
cells at day 0 (i.e., a day before the preprogramming factors) and
incubated for about 14-30 days.

[0185] In other embodiments, different inhibitors are added at different
times. In some embodiments, at 1-7 days after the delivery of the
reprogramming factors, the cells are contacted with compound combination
of an TGFβ receptor/ALK5 inhibitor (e.g., SB431542) and a ROCK
inhibitor for 1-8 days, followed by contacting the cells with the
TGFβ receptor/ALK5 inhibitor, ROCK inhibitor and a MEK/ERK pathway
inhibit (e.g., PD0325901) for 1-8 days. This can be optionally followed
by contact with the TGFβ receptor/ALK5 inhibitor and MEK/ERK pathway
inhibitor (but not necessarily the ROCK inhibitor) for 1-4 days, followed
by contact with the MEK/ERK pathway inhibitor (but not the TGFβ
receptor/ALK5 inhibitor or ROCK inhibitor), and optionally finally with
basal (e.g., basal human) ES medium without inhibitors for 1-4 days.
Other combinations can also be employed.

IV. Transformation

[0186] This invention employs routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of use
in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression:
A Laboratory Manual (1990); and Current Protocols in Molecular Biology
(Ausubel et al., eds., 1994)).

[0187] In some embodiments, the species of cell and protein to be
expressed is the same. For example, if a mouse cell is used, a mouse
ortholog is introduced into the cell. If a human cell is used, a human
ortholog is introduced into the cell.

[0188] It will be appreciated that where two or more proteins are to be
expressed in a cell, one or multiple expression cassettes can be used.
For example, where one expression cassette expresses multiple
polypeptides, a polycistronic expression cassette can be used.

A. Plasmid Vectors

[0189] In certain embodiments, a plasmid vector is contemplated for use to
transform a host cell. In general, plasmid vectors containing replicon
and control sequences which are derived from species compatible with the
host cell are used in connection with these hosts. The vector can carry a
replication site, as well as marking sequences which are capable of
providing phenotypic selection in transformed cells.

B. Viral Vectors

[0190] The ability of certain viruses to infect cells or enter cells via
receptor-mediated endocytosis, and to integrate into host cell genome and
express viral genes stably and efficiently have made them attractive
candidates for the transfer of foreign nucleic acids into cells (e.g.,
mammalian cells). Non-limiting examples of virus vectors that may be used
to deliver a nucleic acid of the present invention are described below.

[0191] i. Adenoviral Vectors

[0192] A particular method for delivery of the nucleic acid involves the
use of an adenovirus expression vector. Although adenovirus vectors are
known to have a low capacity for integration into genomic DNA, this
feature is counterbalanced by the high efficiency of gene transfer
afforded by these vectors. "Adenovirus expression vector" is meant to
include those constructs containing adenovirus sequences sufficient to
(a) support packaging of the construct and (b) to ultimately express a
tissue or cell-specific construct that has been cloned therein. Knowledge
of the genetic organization or adenovirus, a ˜36 kb, linear,
double-stranded DNA virus, allows substitution of large pieces of
adenoviral DNA with foreign sequences up to 7 kb (Grunhaus et al.,
Seminar in Virology, 200(2):535-546, 1992)).

[0193] ii. AAV Vectors

[0194] The nucleic acid may be introduced into the cell using adenovirus
assisted transfection. Increased transfection efficiencies have been
reported in cell systems using adenovirus coupled systems (Kelleher and
Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc Natl Acad Sci
USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64, 1994.).
Adeno-associated virus (AAV) is an attractive vector system as it has a
high frequency of integration and it can infect non-dividing cells, thus
making it useful for delivery of genes into mammalian cells, for example,
in tissue culture (Muzyczka, Curr Top Microbiol Immunol, 158:97-129,
1992) or in vivo. Details concerning the generation and use of rAAV
vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each
incorporated herein by reference.

[0195] iii. Retroviral Vectors

[0196] Retroviruses have promise as gene delivery vectors due to their
ability to integrate their genes into the host genome, transferring a
large amount of foreign genetic material, infecting a broad spectrum of
species and cell types and of being packaged in special cell-lines
(Miller et al., Am. J. Clin. Oncol., 15(3):216-221, 1992).

[0197] In order to construct a retroviral vector, a nucleic acid (e.g.,
one encoding gene of interest) is inserted into the viral genome in the
place of certain viral sequences to produce a virus that is
replication-defective. To produce virions, a packaging cell line
containing the gag, pol, and env genes but without the LTR and packaging
components is constructed (Mann et al., Cell, 33:153-159, 1983). When a
recombinant plasmid containing a cDNA, together with the retroviral LTR
and packaging sequences is introduced into a special cell line (e.g., by
calcium phosphate precipitation for example), the packaging sequence
allows the RNA transcript of the recombinant plasmid to be packaged into
viral particles, which are then secreted into the culture media (Nicolas
and Rubinstein, In: Vectors: A survey of molecular cloning vectors and
their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp.
494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York:
Plenum Press, pp. 149-188, 1986; Mann et al., Cell, 33:153-159, 1983).
The media containing the recombinant retroviruses is then collected,
optionally concentrated, and used for gene transfer. Retroviral vectors
are able to infect a broad variety of cell types. However, integration
and stable expression typically involves the division of host cells
(Paskind et al., Virology, 67:242-248, 1975).

[0198] Lentiviruses are complex retroviruses, which, in addition to the
common retroviral genes gag, pol, and env, contain other genes with
regulatory or structural function. Lentiviral vectors are well known in
the art (see, for example, Naldini et al., Science, 272(5259):263-267,
1996; Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer et
al., J Virol., 71(9):6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and
5,994,136). Some examples of lentivirus include the Human
Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency
Virus: SIV. Lentiviral vectors have been generated by multiply
attenuating the HIV virulence genes, for example, the genes env, vif,
vpr, vpu and nef are deleted making the vector biologically safe.

[0199] Recombinant lentiviral vectors are capable of infecting
non-dividing cells and can be used for both in vivo and ex vivo gene
transfer and expression of nucleic acid sequences. For example,
recombinant lentivirus capable of infecting a non-dividing cell wherein a
suitable host cell is transfected with two or more vectors carrying the
packaging functions, namely gag, pol and env, as well as rev and tat is
described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
One may target the recombinant virus by linkage of the envelope protein
with an antibody or a particular ligand for targeting to a receptor of a
particular cell-type. By inserting a sequence (including a regulatory
region) of interest into the viral vector, along with another gene which
encodes the ligand for a receptor on a specific target cell, for example,
the vector is now target-specific.

[0200] iv. Delivery using Modified Viruses

[0201] A nucleic acid to be delivered may be housed within an infective
virus that has been engineered to express a specific binding ligand. The
virus particle will thus bind specifically to the cognate receptors of
the target cell and deliver the contents to the cell. A novel approach
designed to allow specific targeting of retrovirus vectors was developed
based on the chemical modification of a retrovirus by the chemical
addition of lactose residues to the viral envelope. This modification can
permit the specific infection of hepatocytes via sialoglycoprotein
receptors.

[0202] Another approach to targeting of recombinant retroviruses was
designed in which biotinylated antibodies against a retroviral envelope
protein and against a specific cell receptor were used. The antibodies
were coupled via the biotin components by using streptavidin (Roux et
al., Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989). Using antibodies
against major histocompatibility complex class I and class II antigens,
they demonstrated the infection of a variety of human cells that bore
those surface antigens with an ecotropic virus in vitro (Roux et al.,
1989).

[0204] The present invention provides for mixtures that improve the
efficiency of generation of iPSCs For example, the invention provides for
mixtures of a TGFβ receptor/ALK5 inhibitor, a MEK/ERK pathway
inhibitor, a Rho GTPase/ROCK inhibitor, in particular embodiments, with
mammalian cells. For example, the mixtures can be included in cell
culture media, with or without cells. The contents of cell culture media
are generally known in the art. Exemplary cell culture media are
described in detail in the Examples. Generally, cell cultures comprising
mammalian cells and agents of the invention (TGFβ receptor/ALK5
inhibitor, a MEK/ERK pathway inhibitor, and a Rho GTPase/ROCK inhibitor)
will initially contain all or substantially all non-pluripotent cells.
However, over time, especially under the conditions of the protocols
described here, a portion of the cells will become pluripotent (i.e.,
iPSCs).

[0205] Cells to be induced to pluripotency can be cultured according to
any method known in the art. General guidelines for culture conditions to
generate iPSCs can be found in, e.g., Maherali, et al., Cell Stem Cell
3:595-605 (2008).

[0206] In some embodiments, the cells are cultured in contact with feeder
cells. Exemplary feeder cells include, but are not limited to fibroblast
cells, e.g., mouse embryonic fibroblast (MEF) cells. Methods of culturing
cells on feeder cells are known in the art.

[0207] In some embodiments, the cells are cultured in the absence of
feeder cells. Cells, for example, can be attached directly to a solid
culture surface (e.g., a culture plate), e.g., via a molecular tether.
The inventors have found that culturing cells induced to pluripotency
have a much greater efficiency of induction to pluripotency (i.e., a
greater portion of cells achieve pluripotency) when the cells are
attached directly to the solid culturing surface compared the efficiency
of otherwise identically-treated cells that are cultured on feeder cells.
Exemplary molecular tethers include, but are not limited to,
Matrigel®, an extracellular matrix (ECM), ECM analogs, laminin,
fibronectin, or collagen. Those of skill in the art however will
recognize that this is a non-limiting list and that other molecules can
be used to attach cells to a solid surface. Methods for initial
attachment of the tethers to the solid surface are known in the art.

[0208] As described herein, in some embodiments, the mixtures of the
invention can include or exclude mammalian cells (including pluripotent
or non-pluripotent cells), and one or more of a HDAC inhibitor, GSK3
inhibitor, or an L-type Ca channel agonist; an activator of the cAMP
pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptor
ligand, e.g., as described in PCT WO/2009/117439.

VIII. Kits

[0209] The present invention also provides kits, e.g., for use in
generating induced pluripotent stem cells. Such kits can comprise any or
all of the reagents described herein, including but not limited to: a
TGFβ receptor/ALK5 inhibitor, a MEK/ERK pathway inhibitor, and/or a
Rho GTPase/ROCK inhibitor, as described herein. These three agents, or
subsets thereof, can be present in the kit in separate vials, or together
as a mixture. The kits of the invention can also include, one or more of
an HDAC inhibitor, a GSK3 inhibitor, or an L-type Ca channel agonist; an
activator of the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor;
and a nuclear receptor ligand.

[0210] In one embodiment, the kits of the invention will include one or
more types of mammalian (e.g., human, mouse, rat, etc.) cells and/or cell
culture media.

[0211] In a particular embodiment, the kits of the invention will include
one or more polynucleotides comprising expression cassettes for
expression of one or more of Oct polypeptide, a Klf polypeptide, a Myc
polypeptide, and a Sox polypeptide. In addition, or alternatively, the
kits can comprise one or more isolated transcription factor proteins,
e.g., one, two, three or all four of an Oct polypeptide, a Klf
polypeptide, a Myc polypeptide, and a Sox polypeptide. In another
particular embodiment, the transcription factor proteins can be fused to
a polypeptide sequence for enhancing transport of the transcription
factor proteins across cell membranes.

VI. Uses for Pluripotent Cells

[0212] The present invention allows for the further study and development
of stem cell technologies, including but not limited to, prophylactic or
therapeutic uses. For example, in some embodiments, cells of the
invention (either pluripotent cells or cells induced to differentiate
along a desired cell fate) are introduced into individuals in need
thereof, including but not limited to, individuals in need of
regeneration of an organ, tissue, or cell type. In some embodiments, the
cells are originally obtained in a biopsy from an individual; induced
into pluripotency as described herein, optionally induced to
differentiate (for examples into a particular desired progenitor cell)
and then transplanted back into the individual. In some embodiments, the
cells are genetically modified prior to their introduction into the
individual.

[0216] In one embodiment, iPSCs can be used in various assays and screen
to identify molecules that modulate their function, including but not
limited to promoting iPSC survival and/or differentiation.

EXAMPLES

[0217] The following examples are offered to illustrate, but not to limit
the claimed invention.

[0219] We tested known inhibitors of the TGFβ receptor and MEK on
1×104 (Feng, B. et al., Cell Stem Cell 4, 301-12 (2009)) human
primary fibroblasts (CRL2097 or BJ) that were retrovirally transduced
with the 4TFs, for their effect on reprogramming kinetics and efficiency
(see FIG. 1A for details). On day 7 (D7) post-infection, the compounds
were added, individually or in combinations, and the cultures were
examined for iPSCs over the next 1-3 weeks.

[0220] On day 7 post-treatment (D14), we observed the strongest effect in
the cultures treated with a combination of ALK5 inhibitor SB431542 (2
μM) and MEK inhibitor PD0325901 (0.5 μM), which resulted in
∥45 large ALP.sup.+ colonies (FIG. 1B) with characteristic
hESC-like morphology, of which over 24 colonies were TRA-1-81.sup.+ (FIG.
1d), and about 6-10 colonies stained positive for SSEA4 and NANOG, a
mature pluripotency factor that is not ectopically introduced (FIGS. 1e
and 1f). Moreover, the treated cultures showed high level expression of
endogenous mRNA for the pluripotency genes (FIG. 1c). In contrast, no
NANOG.sup.+ colonies were observed in the untreated control cultures
(FIG. 1E & FIG. 4A) or in cultures that were treated with PD0325901 alone
(FIG. 4A). However, in the cultures treated with only SB431542, we still
observed 1-2 ALP.sup.+ hESC-like colonies (FIG. 4A). Importantly, the
combined effect of both the inhibitors (FIGS. 4b & 4c), as well as the
individual effect of SB431542 was dose dependent.

[0221] When we maintained the SB431542 plus PD0325901 treated cultures for
30 days without splitting, we obtained about 135 iPSC colonies per well
(FIG. 2D), a >100 fold improvement in efficiency over the conventional
method. Consistent with previous reports (Takahashi, K. et al., Cell 131,
861-72 (2007)), in untreated controls carrying 4TFs, we observed 1-2 iPSC
colonies in addition to several granulate colonies (FIG. 2c). These
granulate structures have been suggested to be partially reprogrammed
colonies (Takahashi, K. et al., Cell 131, 861-72 (2007)). We also
observed granulate colonies in the SB431542 treated cultures, which
outnumbered by several fold the few hESC-like colonies. Interestingly,
the number of granulate colonies was dramatically reduced in the combined
SB431542 and PD0325901 treatment, which resulted in a concomitant
increase in the number of hESC-like colonies. This suggested that a
combined inhibition of ALK5 and MEK may guide partially reprogrammed
colonies to a fully reprogrammed state and thereby improve the overall
reprogramming process. Moreover, the fact that we observed improved
induction of iPSCs as early as 7 days post-treatment suggests that
treatment with these small molecules not only improved the efficiency of
the reprogramming process but may also have accelerated its kinetics
(FIG. 1A). Additional experiments are required to determine whether the
reprogrammed cells at this stage indeed become fully independent of
exogenous reprogramming factors earlier than in untreated cultures.

[0222] Although iPSC colonies were picked and expanded, as in hESC
cultures, the cultures split by trypsinization resulted in poor survival.
From a recent screen performed in our laboratory we identified a novel
small molecule, Thiazovivin (FIG. 5), which dramatically improved the
survival of hESCs upon trypsinization. Addition of Thiazovivin to our
cocktail of SB431542 and PD0325901 also vastly improved the survival of
iPSCs after splitting by trypsinization (FIG. 2A), and a large number of
reprogrammed colonies were obtained. From 10,000 cells that were
originally seeded, a single 1:4 splitting on day 14 resulted in
˜1,000 hESC-like colonies on day 30 (FIG. 2E), while two rounds of
splitting (on day 14 and on day 21 (1:10)) resulted in ˜11,000
hESC-like colonies (FIGS. 2c & 2e) on day 30. These colonies showed high
levels of endogenous mRNA (FIG. 2F) and protein expression (FIGS. 2b &
2c) of pluripotency markers, while the expression of the four transgenes
could hardly be detected (FIG. 2F). In contrast, no iPSC colonies were
obtained from untreated or 2 compound-treated samples that were
trypsinized (Table 1).

[0223] To examine whether the positive effect of Thiazovivin is solely due
to survival of colonies after splitting or whether it also augments the
reprogramming effect of combined SB431542 and PD0325901 treatment, we
tested the 3 compound cocktail on 4TF-transduced cells that were not
subjected to splitting. In these cultures, by day 14 we observed
˜25 large colonies that were all expressing Nanog (FIG. 1E). By day
30 we observed ˜205 very large NANOG.sup.+ colonies (FIG. 2D), that
were also TRA-1-81.sup.+ and SSEA4.sup.+ (data not shown), which
translated to a more than 200 fold improvement in efficiency over no
compound treatment, and a two-fold increase over 2 compound treatment.

[0224] Two compound treatment also resulted in a larger number of alkaline
phosphatase-positive colonies compared to untreated controls when the
reprogramming factors were introduced using a lentiviral, rather than a
retroviral system (FIG. 6A). Furthermore, the 3 compound cocktail did not
appear to influence reprogramming factor expression from retroviral
vectors (FIG. 6B-f).

[0225] The iPSC colonies generated using the 3 compound cocktail were
readily and stably expanded for long term under conventional hESC culture
conditions (over 20 passages) and they closely resembled hESCs in terms
of morphology, typical pluripotency marker expression and differentiation
potentials. They exhibited a normal karyotype (FIG. 7) and could be
differentiated into derivatives of all three germ layers, both in vitro
(FIGS. 3a & 3b) and in vivo (FIG. 3c). These results also suggested that
there is no short term adverse effect associated with the much more
convenient trypsinization procedure.

[0226] The demonstration that TGFβ and MEK-ERK pathway inhibition
improved fibroblast reprogramming suggested critical roles for these two
signaling pathways and MET mechanisms in the process. Consistently,
addition of TGFβ had an inhibitory effect on 4 factor-mediated
reprogramming of fibroblasts (data not shown). TGFβ and its family
members play important contextual roles in self-renewal and
differentiation of ESCs (Watabe, T. and Miyazono, K., Cell Res. 19,
103-15 (2009)). Moreover, TGFβ is a prototypical cytokine for
induction of epithelial mesenchymal transition (EMT) and maintenance of
the mesenchymal state (Willis, B. C. and Borok, Z., Am. J. Physiol. Lung
Cell Mol. Physiol.293, L525-34 (2007)). A major end point of this
signaling, in this context, is down regulation of E-cadherin (Thiery, J.
P. and Sleeman, J. P., Nat. Rev. Mol. Cell Biol., 7, 131-42 (2006)).
E-cadherin has been shown to be important for the maintenance of
pluripotency of ESCs and has been recently suggested to be a regulator of
NANOG expression (Chou, Y. F. et al., Cell 135, 449-61 (2008)). Therefore
inhibition of TGFβ signaling, which results in de-repression of
epithelial fate, could benefit the reprogramming process in multiple
ways. ERK signaling also promotes EMT (Thiery, J. P. and Sleeman, J. P.,
Nat. Rev. Mol. Cell Biol. 7, 131-42 (2006)), and is downstream of
TGFβ in the process (Chou, Y. F. et al., Cell 135, 449-61 (2008)).
We had previously shown that the effect of reversine, a small molecule
which can reprogram myoblasts to a multipotent state, is mediated in part
through inhibition of MEK-ERK (Chen, S. et al., Proc. Natl. Acad. Sci.
USA 104, 10482-87 (2007)). This may explain the effect observed in
reprogramming when it was combined with TGFβ inhibition.

[0227] The chemical platform described here is unique, in that it
modulates upstream signaling pathways and could radically improve
reprogramming on a general cell type, like fibroblasts. The chemical
conditions described here provide a basic platform for non-viral and
non-DNA based (Zhou, H. et al., Cell Stem Cell 4, 381-84, (2009)), more
efficient and safer reprogramming methods, which could yield an unlimited
supply of safe human iPSCs for various applications.

[0230] Lentiviruses carrying OCT4, NANOG, SOX2 & LIN28 were produced as
described before (Yu, J. et al., Science 318, 1917-20 (2007)). For
retrovirus production, PLAT-E packaging cells were plated at
1×106 cells/well of a 6-well plate. After 24 hours, the cells
were transfected with pMXs vectors carrying OCT4, SOX2, c-MYC and KLF4
cDNAs using Fugene 6 transfection reagent (Roche) according to
manufacturer's instructions. Twenty-four hours after transfection, the
medium was replaced with fresh medium and the plate was transferred to
32° C. for retrovirus production. The viruses were collected at 48
hours and 72 hours, and filtered with 0.45 μm filter before
transduction.

[0231] The Slc7a1-expressing human fibroblast cells were seeded at
1×105 cells/well of a 6 well plate on the day 1. On day 2,
0.25 ml of each retroviral supernatant was added to the cells in the
presence of 6 μg/ml polybrene. A second round of transduction was done
on day 3. Infection efficiency was estimated by fluorescence microscopy
on cells transduced in parallel with GFP or RFP gene-carrying
retroviruses. Seven days after initial transduction, fibroblasts were
harvested by trypsinization and re-plated at 1×104 cells/well
of a 6 well plate coated with matrigel (1:50 dilution, cat 354234, BD
Biosciences). For compound treatment, the cells were cultured in human
reprogramming medium (DMEM/F12, 20% Knockout serum replacer, 1× MEM
Non-Essential amino acid, 1× glutamax, 0.11 mM 2-Mercaptoethanol,
20 ng/ml bFGF and 1,000 U/ml LIF) and were treated with 2 μM SB431542
(Stemgent), 0.5 μM PD0325901 (Stemgent), 0.5 μM Thiazovivin, or
combinations of the compounds. The media were changed every 2-3 days
depending on the cell density. Seven days after compound treatment,
either the plates were fixed and stained for Alkaline phosphatase (ALP)
activity, or stained for protein markers, or the cultures were continued
with or without indicated splitting by trypsinization till day 30. For
split cultures, the cells were split (1:4) and re-plated onto irradiated
CF-1 MEF feeder layer (2.5×105 cells/well) in each well of 6 well
plate and were split (1:10) again on day 21. The cells were maintained in
the same media and compound cocktail described above except for the
concentrations of PD0325901 (0.5 μM for D14 and 1 μM for D21) and
SB431542 (0.5-1 μM after D14). The iPSC colonies were subsequently
maintained in conventional hESC media in the absence of the above
compounds.

[0233] Generation of embryoid bodies and in vitro differentiation were
performed as described elsewhere (Takahashi, K. et al., Cell 131, 861-72
(2007)). For the teratoma assay, 3-5 million cells were injected under
the kidney capsule of SCID mice. Thirty one days later the tumors were
excised and fixed in 4% paraformaldehyde and histologically analyzed at
the TSRI histology core facility. The use of SCID mice was approved by
the UCSD animal research committee.

RT-PCR

[0234] Total RNA was extracted from cells using RNeasy minikit (Qiagen).
cDNAs were synthesized according to product instructions using
superscript III first strand synthesis kit (Invitrogen). Two microliters
of the reaction product was used for 24-28 PCR cycles using respective
primers. The sequences of the primers are described elsewhere (Takahashi,
K. et al., Cell 131, 861-72 (2007)).

Flow Cytometry

[0235] For flow cytometry analysis, the cultures were mildly trypsinized
and harvested from 6 well plates. The cells were washed and resuspended
in FACS buffer (PBS, 2 mM EDTA, 2 mM HEPES, 1% FBS), and were analyzed on
a FACS Calibur cytometer (Becton Dickinson, San Jose, Calif.) with the
CellQuest program.

[0237] Here we report a novel small molecule cocktail that enables
reprogramming of human primary somatic cells to iPSCs with exogenous
expression of only OCT4.

[0238] Among several readily available primary human somatic cell types,
keratinocytes that can be easily isolated from human skin or hair
follicle represent an attractive cell source for reprogramming, because
they endogenously express KLF4 and cMYC, and were reported to be
reprogrammed more efficiently using the conventional four TFs or three
TFs (without MYC) (Aasen, T. et al., Nat Biotechnol 26:1276-1284 (2008);
Maherali, N. et al., Cell Stem Cell 3, 340-345(2008)). More recently, we
reported that dual inhibition of TGFβ and MAPK/ERK pathways using
small molecules (i.e. SB431542 and PD0325901, respectively) provides a
drastically enhanced condition for reprogramming of human fibroblasts
with four exogenous TFs (i.e. OSKM) (Lin, T. et al., Nat Methods
6:805-808 (2009)). Furthermore, we have shown that such dual pathway
inhibition could also enhance reprogramming of human keratinocytes by two
exogenous TFs (i.e. OK) with two small molecules, Parnate (an inhibitor
of lysine-specific demethylase 1) and CHIR99021 (a GSK3 inhibitor) (Li,
W. et al., Stem Cells 27:2992-3000 (2009)). However, such 2-TFs
reprogramming process was very inefficient and complex (e.g. involving
two exogenous TFs and four chemicals), and reprogramming with even one
less TF appeared daunting. Toward the OCT4 only reprogramming, we
developed a step-wise strategy in refining reprogramming condition and
identifying new reprogramming chemical entities. We first attempted to
further optimize the reprogramming process under the four or three TFs
(i.e. OSKM or OSK) condition in neonatal human epidermal keratinocytes
(NHEKs) by testing various inhibitors of TGFβ and MAPK pathways at
different concentrations using previously reported human iPSC
characterization methods (Lin, T. et al., Nat Methods 6:805-808 (2009)).
Encouragingly, we found that the combination of 0.5 μM PD0325901 and
0.5 μM A-83-01 (a more potent and selective TGFβ receptor
inhibitor) was more effective in enhancing reprogramming of human
keratinocytes transduced with OSKM or OSK (FIG. 8A). Remarkably, when we
further reduced viral transductions to only two factors/OK, we could
still generate iPSCs from NHEKs when they were treated with 0.5 μM
PD0325901 and 0.5 μM A-83-01, although with low efficiency. Then we
began screening additional small molecules from a collection of known
bioactive compounds at various concentrations as previously reported.
Among dozens of compounds tested so far, surprisingly we found that a
small molecule activator of PDK1 (3'-phosphoinositide-dependent
kinase-1), PS48 (5 μM) that has never been reported in reprogramming,
can significantly enhance the reprogramming efficiency about fifteen
fold. Interestingly, we also found that 0.25 mM sodium butyrate (NaB, a
histone deacetylase inhibitor) turned out to be much more reliable and
efficient than the previously reported 0.5 mM VPA for the generation of
iPSCs under OK condition (FIG. 8B). Subsequent follow-up studies
demonstrated that combination of 5 μM PS48 and 0.25 mM NaB could
further enhance the reprogramming efficiency over twenty-five fold (FIG.
8b and Table 4). With such unprecedented efficiency in reprogramming
NHEKs under only two TFs, we further explored the possibility of
generating iPSCs with OCT4 alone by refining combinations of those small
molecules during different treatment windows. Primary NHEKs were
transduced with OCT4 and treated with chemicals (FIG. 8c). Among various
conditions, small iPSC colonies resembling hESCs (four to six colonies
out of 1,000,000 seeded cells) appeared in OCT4 infected NHEKs that were
treated with 0.25 mM NaB, 5 μM PS48 and 0.5 μM A-83-01 during the
first four weeks, followed by treatment with 0.25 mM NaB, 5 μM PS48,
0.5 μM A-83-01 and 0.5 μM PD0325901 for another four weeks (FIG.
8c). Such TRA-1-81 positive iPSC colonies (FIG. 8D) grew larger under
conventional hESC culture media and could be serially passaged to yield
stable iPSC clones that were further characterized (FIGS. 8e and 9). More
significantly, OCT4 only iPSCs could also be generated from human adult
keratinocytes by addition of 2 μM Parnate and 3 μM CHIR99021 (which
had been shown to improve reprogramming of NHEKs under OK condition) to
this chemical cocktail. After the reliable reprogramming of primary
keratinocytes to iPSCs by OCT4 and small molecules, we further applied
the conditions to other human primary cell types, including HUVECs
(differentiated mesoderm cells) and AFDCs (amniotic fluid derived cells).
Similarly, TRA-1-81 positive iPSC colonies appeared in OCT4 infected
HUVECs and AFDCs that were treated with chemicals. Remarkably, it
appeared that reprogramming of HUVECs and AFDCs was more efficient and
faster than reprogramming of NHEKs under the OCT4 and small molecule
conditions (Table 4). Two clones of iPSCs from each cell type were
long-term expanded for over 20 passages under conventional hESC culture
condition and further characterized (Table 5).

[0239] These stably expanded hiPSC-OK and hiPSC-O cells are
morphologically indistinguishable to hESCs, and could be cultured on
ECM-coated surface under feeder-free and chemically defined conditions
(FIG. 8e and FIG. 13). They stained positive for alkaline phosphatase
(ALP) and expressed typical pluripotency markers, including OCT4, SOX2,
NANOG, TRA-1-81 and SSEA4, detected by immunocytochemistry/ICC (FIG. 8e,
10b, FIGS. 11-12). In addition, RT-PCR analysis confirmed the expression
of the endogenous human OCT4, SOX2, NANOG, REX1, UTF1, TDGF2, FGF4 genes,
and silencing of exogenous OCT4 and KLF4 (FIGS. 9a and 10c). Furthermore,
bisulfite sequencing analysis revealed that the OCT4 and NANOG promoters
of hiPSC-OK and hiPSC-O cells are largely demethylated (FIGS. 9b and
10d). This result provides further evidence for reactivation of the
pluripotency transcription program in the hiPSC-OK and hiPSC-O cells.
Global gene expression analysis of hiPSC-O cells, NHEKs and hESCs showed
that hiPSC-O cells are distinct from NHEKs (Pearson correlation value:
0.87) and most similar to hESCs (Pearson correlation value: 0.98) (FIG.
9c). Genetyping analysis showed that hiPSC-O cells only contained the
OCT4 transgene without the contamination of transgene KLF4 or SOX2 (FIG.
15). Southern blot analysis showed that there were multiple different
integration sites of the OCT4 transgene (FIG. 16) among different clones.
In addition, karyotyping result demonstrated that hiPSC-O maintained
normal karyotype during the whole reprogramming and expansion process
(FIG. 17). Furthermore, DNA fingerprinting test excluded the possibility
that these hiPSCs arose from hESC contamination in the laboratory (Table
6). To examine the developmental potential of these hiPSC-O cells, they
were differentiated in vitro by the standard embryoid body (EB)
differentiation method. ICC analyses demonstrated that they could
effectively differentiate into βIII-tubulin.sup.+ characteristic
neuronal cells (ectoderm), SMA.sup.+ mesodermal cells, and AFP.sup.+
endodermal cells (FIGS. 9d and 10e). Quantitative PCR analyses further
confirmed the expression of these and additional lineage specific marker
genes, including ectodermal cells (βIII-tubulin and NESTIN),
mesodermal cells (MSX1 and MLC2a), and endodermal cells (FOXA2 and AFP)
(FIG. 9e). Following EB protocol, these hiPSC-OK and hiPSC-O cells could
also give rise to rhythmically beating cardiomyocytes. To test their in
vivo pluripotency, they were transplanted into SCID mice. Four-six weeks
later, these hiPSC-O cells effectively generated typical teratomas
containing derivatives of all three germ layers (FIGS. 9f and 10f).
Collectively, these in vitro and in vivo characterizations demonstrated
that a single transcription factor, OCT4, combined with a defined small
molecule cocktail is sufficient to reprogram several human primary
somatic cells to iPSCs that are morphologically, molecularly and
functionally similar to pluripotent hESCs.

[0240] The studies presented above have a number of important
implications: (1) Although fetal NSCs were shown to be reprogrammed to
iPSCs by ectopic expression of Oct4 alone, there have been significant
skepticisms around whether exogenous Oct4 gene alone would be sufficient
to reprogram other more practical human somatic cells that do not
endogenously express Sox2 (one of the two master pluripotency genes in
reprogramming), are at later developmental stages (e.g. early
embryonic/fetal vs. born/adult), and can be obtained without significant
harms to the individual. To our knowledge, our study is the first
demonstration that iPSCs can be practically derived from readily
available primary human somatic cells (e.g. keratinocytes) transduced
with a single exogenous reprogramming gene, Oct4. In contrast to neural
stem cells from the brain, keratinocytes are more accessible and can be
easily obtained from born individuals with less invasive procedures. This
further strengthens the strategy of exploiting various practically
accessible human somatic cells for iPSC generation with safer approaches
and/or better qualities. Thus, this new method and its further
development would significantly facilitate production of patient-specific
pluripotent stem cells for various applications. (2) Although small
molecules and their combinations have been identified to replace only one
or two reprogramming TFs, it becomes exponentially challenging to
generate iPSCs when more exogenous reprogramming TFs are omitted
together. The identification of this new small molecule cocktail, which
functionally re-places three master transcription factors all together
(i.e. Sox2, Klf4 and cMyc) in enabling generation of iPSCs with Oct4
alone, represents another major step toward the ultimate reprogramming
with only small molecules, and further proved and solidified the chemical
approach to iPSCs. (3) This demonstrated single gene condition also has a
significant implication for protein-induced pluripotent stem cell (piPSC)
technology. A practical challenge for piPSC technology is large-scale and
reliable production of the four transducible reprogramming proteins, each
of which behaves differently in manufacture (e.g. their expression,
folding, stability etc.). Clearly, combining this small molecule cocktail
with a single transducible protein would significantly simplify the piPSC
technology and facilitate its applications. (4) More significantly, we
identified a new small molecule, PS48, with a new target/mechanism in
enhancing reprogramming. PS48 is an allosteric small molecule activator
of PDK1, which is an important upstream kinase for several AGC kinases,
including Akt/PKB (Alessi et al., Curr Biol 7, 261-269 (1997)). Its
reprogramming enhancing effect may be partly attributed to the activation
of Akt/PKB, which promotes cell proliferation and survival (Manning, B.
D., Cantley, L. C., Cell 129, 1261-1274 (2007)). Further in-depth
characterizations on how PDK1-involved mechanisms are precisely regulated
during reprogramming process should provide additional insights
underlying reprogramming and pluripotency. Furthermore, because there
might be even greater hidden risks (e.g. more subtle genetic and/or
epigenetic abnormalities could be generated or selected during the
reprogramming process) imposed by the low efficiency and slow kinetics of
reprogramming, identification of new small molecules for enhancing
reprogramming as illustrated again in this study would always be highly
valuable toward a safer, easier and more efficient procedure for human
iPSC generation. (5) Finally, this new and powerful small molecule
cocktail for reprogramming validated the step-wise chemical optimization
and screening strategy presented here as a productive approach toward the
ultimate purely chemical-induced pluripotent stem cells. Moreover, the
finding that different small molecules modulating the same
target/mechanism could have significantly different effects on
reprogramming in a different context, exemplified by A-83-01's and NaB's
better reprogramming enhancing activities in human keratinocytes,
suggests the importance of "individualized" optimization and treatment
with different regimens for specific reprogramming context.

[0243] NHEKs were cultured in a 100 mm tissue culture dish and transduced
3 times (3-4 hours each transduction) with freshly produced lentivirus
supernatants. 1,000,000 transduced NHEKs were seeded on the irradiated
x-ray inactivated CF1 MEF feeder cells in a 100-mm dish and cultured in
KCM and treated with 5 μM PS48, 0.25 mM NaB (Stemgent) and 0.5 μM
A-83-01 (Stemgent) for 2 weeks, followed by changing half volume of media
to hESCM and supplementing with 5 μM PS48, 0.25 mM NaB and 0.5 μM
A-83-01 for another 2 weeks. Then cell culture media were changed to
hESCM and supplemented with 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01
and 0.5 μM PD0325901 (Stemgent) for additional four weeks. The same
OCT4 infected keratinocytes cultured in media without chemicals were used
as a control. The culture was split by Accutase (Millipore) and treated
with 1 μM Thiazovivin (Stemgent) in the first day after splitting. The
iPSC colonies stained positive by Alexa Fluor 555 Mouse anti-Human
TRA-1-81 antibody (BD Pharmingen) were picked up for expansion on feeder
cells in hESCM and cultured routinely.

Reprogramming of HUVECs

[0244] HUVECs were cultured in a 100 mm tissue culture dish and transduced
2 times (4-6 hours each transduction) with freshly produced lentivirus
supernatants. 200,000 transduced HUVECs were seeded on gelatin coated
100-mm dish, cultured in HCM, and treated with 5 μM PS48, 0.25 mM NaB
and 0.5 μM A-83-01 for 2 weeks, followed by changing half volume of
media to hESCM and supplementing with 5 μM PS48, 0.25 mM NaB and 0.5
μM A-83-01 for another 2 weeks. Then cell culture media were changed
to hESCM and supplemented with 5 μM PS48, 0.25 mM NaB, 0.5 μM
A-83-01 and 0.5 μM PD0325901 for additional 1-2 weeks. The iPSC
colonies stained positive by Alexa Fluor 555 Mouse anti-Human TRA-1-81
antibody were picked up for expansion on feeder cells in hESCM and
cultured routinely. The culture was split by Accutase and treated with 1
μM Thiazovivin in the first day after splitting.

In Vitro Differentiation

[0245] The in vitro differentiation of hiPSCs was carried out by the
standard embryoid body (EB) method. Briefly, the hiPSCs were dissociated
by Accutase (Millipore), cultured in ultra-low attachment 6-well plate
for eight days and then transferred to Matrigel-coated E-well plate in
differentiation medium. The cells were fixed for immunocytochemical
analysis or harvested for RT-PCR tests eight days later. Differentiation
medium: DMEM/F 12, 10% FBS, 1% Glutamax, 1% Non-essential amino acids, 1%
penicillin/streptomycin, 0.1 mM β-mercaptoethanol.

Alkaline Phosphatase Staining and Immunocytochemistry Assay

[0246] Alkaline Phosphatase staining was performed according to the
manufacturer's protocol using the Alkaline Phosphatase Detection Kit
(Stemgent). Standard immunocytochemistry assay was carried out as
previously reported (Li, W. et al., Stem Cells 27:2992-3000 (2009)).
Primary antibodies used can be found in the Table 3. Secondary antibodies
were Alexa Fluor 488 donkey anti-mouse or anti-rabbit IgG (1:1000)
(Invitrogen). Nuclei were visualized by DAPI (Sigma-Aldrich) staining.
Images were captured using a Nikon Eclipse TE2000-U microscope.

Gene Expression Analysis by RT-PCR and qRT-PCR

[0247] For RT-PCR and qRT-PCR analysis, total RNA was extracted from human
iPSCs using the RNeasy Plus Mini Kit in combination with QIAshredder
(Qiagen). First strand reverse transcription was performed with 2 μg
RNA using iScript® cDNA Synthesis Kit (BioRad). The expression of
pluripotency markers was analyzed by RT-PCR using Platinum PCR SuperMix
(Invitrogen). The expression of lineage specific markers after
differentiation was analyzed by qRT-PCR using iQ SYBR Green Supermix
(Bio-Rad). The primers can be found in the Table 2.

Microarray Analysis

[0248] The Human Ref-8_v3 expression Beadchip (Illumina, CA, USA) was used
for microarray hybridizations to examine the global gene expression of
NHEKs, hiPSC and hES cells. Biotin-16-UTP-labeled cRNA was synthesized
from 500 ng total RNA with the Illumina TotalPrep RNA amplification kit
(Ambion AMIL1791, Foster City, Calif., USA). The hybridization mix
containing 750 ng of labeled amplified cRNA was prepared according to the
Illumina BeadStation 500× System Manual (Illumina, San Diego,
Calif., USA) using the supplied reagents and GE Healthcare
Streptavidin-Cy3 staining solution. Hybridization to the Illumina Human
Ref-8_v3 expression Beadchip was for 18 h at 55° C. on a BeadChip
Hyb Wheel. The array was scanned using the Illumina BeadArray Reader. All
samples were prepared in two biological replicates. Processing and
analysis of the microarray data were performed with the Illumina
BeadStudio software. The data were subtracted for background and
normalized using the rank invariant option.

Bisulfate Genomic Sequencing

[0249] Genomic DNAs were isolated using the Non Organic DNA Isolation Kit
(Millipore) and then treated with the EZ DNA Methylation-Gold Kit (Zymo
Research Corp., Orange, Calif.). The treated DNAs were then used as
templates to amplify sequences of interest. Primers used for OCT4 and
NANOG promoter fragment amplification are indicated in Table 2. The
resulting fragments were cloned using the TOPO TA Cloning Kit for
sequencing (Invitrogen) and sequenced.

[0251] The hiPSC lines were harvested by using 0.05% Trypsin-EDTA. Five
million cells were injected under the kidney capsule of SCID mice (n=3).
After 4-6 weeks, well developed teratomas were harvested, fixed and then
histologically analyzed at TSRI histology core facility.

[0253] Those cell lines characterized were long-term expanded for over 20
passages under conventional hESC culture condition and further
characterized for marker expression and pluripotency; while other cell
lines established were stored at passage 5 or 6. Blank entries indicate
not determined.

[0255] It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in the art
and are to be included within the spirit and purview of this application
and scope of the appended claims. All publications, patents, and patent
applications cited herein are hereby incorporated by reference in their
entirety for all purposes.